RNF115, or Breast Cancer-Associated Gene 2 (BCA2), encodes a RING-finger ubiquitin E3 ligase, expression of which was associated with estrogen receptor (ER)-positive status in human breast tumors. Although the BCA2 promoter contains several estrogen response element (ERE) half-sites, the role of ER in the regulation of BCA2 transcription has not been reported. The aim of this study is to investigate the molecular mechanism by which estrogen regulates BCA2 transcription. BCA2 mRNA and protein levels were examined by RT-PCR and Western blot analysis, respectively, and localization was assessed by immunofluorescence. BCA2 promoter activity in response to E2 was tested by a dual luciferase reporter assay and ER binding to the BCA2 promoter was examined by chromatin immunoprecipitation assay. We found that BCA2 mRNA and protein levels are regulated by estrogen in ER-positive MCF7 breast cancer cells and MDA MB 231 cells stably transfected with ER. Estrogen treatment in hormonal depleted MCF7 and MDA MB 231/ER stably transfected cells resulted in increased nuclear ER and cytoplasmic and nuclear BCA2 staining. Cycloheximide is not able to inhibit BCA2 mRNA levels, suggesting potential BCA2 regulation at the transcriptional level. Anti-estrogens like tamoxifen and ICI 182 178 counteracted E2-induced BCA2 protein and knockdown of ER by ER siRNA resulted in a significant decrease in BCA2 protein and a lower nuclear expression pattern. Estrogen treatment lead to a significant increase in BCA2 promoter response, associated with increased binding of ER to the ERE region of the BCA2 promoter. BCA2 is therefore a newly identified transcriptional target of estrogen receptor.
We will discuss designer stimulants, alpha and baclofen agonist withdrawal, and the clinical entity known as posterior reversible encephalopathy syndrome (PRES). Additionally, we examine the controversial "unopposed alpha" phenomenon which may result from use of beta-adrenergic antagonist in the presence of stimulant toxicity. These topics encompass clinical situations and disease entities that are increasingly encountered and are often either unanticipated or under-recognized.
SUMOylation is a common post-translational modification in which small ubiquitin-like modifier (SUMO) protein moiety is attached to a lysine residue of the target protein and alters its stability, protein-protein interactions, protein-DNA interactions and subcellular localization. Recently, there is an increased recognition for this process as SUMO conjugation plays a critical role in basic cellular processes like proliferation, intracellular signaling and stabilization of transcriptional factors and chromatin remodeling factors. We are studying the Breast Cancer Associated Gene 2 (BCA2), a well characterized ubiquitin E3 ligase which we discovered by subtractive hybridization cloning. In order to identify substrates for the E3 ligase, we performed bacterial two-hybrid screening and found that the SUMO conjugating enzyme Ubc9 as a binding partner. In this study, we examined, whether the BCA2 ligase might be involved in sumoylation.Bioinformatic analyses of BCA2 sequences by SUMOplotTM prediction revealed Lysine 32 as the possible SUMO modification site. To establish direct protein-protein interactions, we performed pull down assays using GST- tagged Ubc9 expressed by the pGEX4T3 expression vector and purified recombinant wild-type BCA2. Results were analyzed by SDS-PAGE Western blotting. To assess if BCA2 and Ubc9 co-localize, we transfected Hek293T cells with BCA2 and Ubc9 constructs and analyzed them using Immunoflorescence staining. We found that BCA2 interacts with Ubc9 and they are co-localized in both the cytoplasm and nucleus. To investigate whether BCA2 is sumoylated by SUMO 1 or SUMO 2/3 we performed in vitro sumoylation assays. SUMO E1 and E2 were incubated along with recombinant BCA2, SUMO1 or SUMO 2/3 respectively for 2 hours at 37°C. The Western blots were probed with SUMO1, SUMO 2/3 and BCA2 antibodies. We also performed in vivo sumoylation with exogenous BCA2 by transfecting pCMV BCA2-Flag vector into Hek293T cells by using Fugene transfection reagent and after 48 hours of transfection, cells were lysed with denaturing lysis buffer and pull downs were performed using anti Flag beads followed by Western blots. These in vitro and in vivo sumoylation studies reveal that BCA2 is sumoylated by SUMO 2/3, but not SUMO1. In the recent past it has become more clear that both the ubiquitination and sumoylation systems communicate and jointly affect the properties of common substrate proteins and there is an increasing evidence in breast cancer development that deregulation of these processes play a vital role. Our findings of a possible involvement of BCA2 in both of these processes opens up avenues that should be further exploited in order to understand the regulatory role of BCA2 in breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4035. doi:10.1158/1538-7445.AM2011-4035
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